This invention relates to a combined scanning probe microscope (SPM) and scanning energy microscope. In specific embodiments, the combination includes an atomic force microscope used for obtaining a two dimensional image of the topography of the surface of a sample, and a confocal laser scanning microscope used for obtaining a two dimensional image of fluorescence emission light or reflected light from the surface of, or a section through, the sample.
Scanning probe microscopy includes the use of an atomic force microscope (AFM), also called a scanning force microscope (SFM), as well as a scanning tunnelling microscope (STM), the former relying on force, the latter on quantum tunneling effects, to image features ranging in size from atoms (0.1 nanometers) to cells (20 micrometers). Both use a feedback system to monitor and control the probe, a mechanical scanning system, usually piezoelectric, to move the sample with respect to the probe in a raster pattern, and a display system that converts the measured data into an image. In an AFM, the probe is a sharp tip mounted on a soft cantilever spring which is brought into contact or close proximity to the surface of the sample. Means are provided to sense the cantilever's deflection. The voltage that a feedback amplifier applies to the piezo is a measure of the height of features on the sample surface. For a general discussion of AFMs see Rugar and Hansma, "Atomic Force Microscopy", Physics Today 43, 23-30 (October, 1990), incorporated herein by reference. Optical detection schemes include beam deflection, for example to a segmented photodiode, and interferometry. Another detection scheme uses a piezoresistive strain sensor embedded in the AFM cantilever. See Tortonese et al., "Atomic Resolution with an Atomic Force Microscope Using Piezoresistive Detection", Appl. Phys. Lett. 62, (8), 834-836 (Feb. 22, 1993), incorporated herein by reference.
AFMs can be operated in liquids as well as in air. See Hansma et al. U.S. Pat. No. Re. 34,489: "Atomic Force Microscope With Optional Replaceable Fluid Cell", incorporated herein by reference. Also, instead of contacting the surface with the cantilever tip during the entire scanning cycle, a tapping mode of operation can be used in which the probe-sample separation is modulated as the probe scans over the sample, causing the probe to tap on the surface only at the extreme of each modulation cycle to therefore minimize frictional forces. See Hansma et al., "Tapping Mode Atomic Force Microscopy in Liquids", Appl. Phys. Lett. 64, (13), 1738-1740 (Mar. 28, 1994), incorporated herein by reference.
AFMs have been used not only for imaging surfaces, but also for manipulating molecules on a surface. The vertical motion of the cantilever tip is detected by sensing the displacement of a reflective beam with a two-segment photodiode. A feedback loop keeps the vertical deflection of the tip, and therefore the force that the tip applies on the surface, constant by moving the surface up and down with an xyz translator. See Weisenhorn et al., "Imaging and Manipulating Molecules on a Zeolite Surface with an Atomic Force Microscope", Science, 247, 1330-1333 (March 1990).
Scanning energy microscopy broadly encompasses any means of scanning with focused energy including confocal scanning optical microscopy (CSOM), such as confocal laser scanning microscopy (CLSM), as well as other focused energy methods such as acoustic microscopes. In CLSM, the specimen is scanned by a diffraction-limited spot of laser light, and light transmitted or reflected by the in-focus illuminated volume element (voxel) of the specimen, or the fluorescence emission excited within it by the incident light, is focused onto a photodetector. An aperture, usually slightly smaller in diameter than the Airy disc image, is positioned in the image plane in front of the detector, at a position confocal with the in-focus voxel of the specimen, which can also be referred to as a focal spot, or specific to a confocal microscope, a confocal spot. Light from the focal spot passes through the aperture of the detector, while light from any region above or below the focal plane is defocused at the aperture plane so that it is largely prevented from reaching the detector, thus contributing essentially nothing to the confocal image. The optical sectioning obtained by reducing out-of-focus blur enables three-dimensional tomography. See Shotton, "Confocal Scanning Optical Microscopy and its Application for Biological Specimens", J. Cell Sci. 94, 175-206 (1989), incorporated herein by reference. Confocal microscopy has been used to measure the profile of a surface. See Hamilton and Wilson, "Surface Profile Measurement Using the Confocal Microscope", J. Appl. Phys. 53 (7), 5320-5322 (July 1982), incorporated herein by reference. For a description of acoustical microscopy, see Quate, "Acoustic Microscopy", Physics Today 38, 34-40 (1985), incorporated herein by reference.
Both scanning probe microscopy and scanning energy microscopy have been successful at imaging biological samples. Since they typically collect different information about the sample, confocal and scanning probe microscopes have been productively combined. An early reference, Engel et al., "Scanning Sensor Microscopy of Biological Membranes", Proceedings of the Xllth International Congress for Electron Microscopy, San Francisco Press, Inc., 108-109 (1990), incorporated herein by reference, describes a scanning sensor microscope combined with a high resolution light microscope equipped for fluorescence and scanning confocal microscopy. Silicon nitride AFM cantilevers were used as well as insulated STM tips and pipettes for ion pickup, each with stationary samples. The combination of a SFM or a STM with a confocal microscope is described in Schabert et al., "Confocal Scanning Laser--Scanning Probe Hybrid Microscope for Biological Applications", Ultramicroscopy 53, 147-157 (1994), incorporated herein by reference. The sample is stationary. The confocal microscope is independent of the SFM or STM and uses galvanometric mirrors to get confocal images using reflection and fluorescence data. A stand-alone AFM combined with a CLSM and used both with the sample in air and under water for simultaneously obtaining AFM and CLSM images is described by Putman et al., "Atomic Force Microscopy Combined with Confocal Laser Scanning Microscopy: a new look at cells", Bioimaging 1, 63-70 (1993), incorporated herein by reference. Here, too, the sample remains stationary and the probes are moved. The combination of an AFM and a simple fluorescent microscope, operated either in air or under liquid, is described by Putman et al., "Polymerized LB Films Imaged with a Combined Atomic Force Microscope-Fluorescence Microscope", Langmuir 8 (10), 3014-3019 (1992), incorporated herein by reference. In Putman (1992), an object can be selected and moved with a translation stage to the AFM tip to be imaged. The sample is stationary during scanning operations. A similar device is described by Henderson et al., "Imaging F-Actin in Fixed Glial Cells with a Combined Optical Fluorescence/Atomic Force Microscope, Neuroimage, 1145-1501 (1993), incorporated herein by reference.
In prior combined AFM-CLSM devices, the AFM is usually a "stand-alone" design in which the confocal (illuminated and detected) spot sweeps through the sample; the sample is not scanned. One drawback to this approach is that the means for scanning the in-focus voxel is independent from the means for scanning the AFM probe. The means for scanning the in-focus voxel, in general, has a different scan range and different nonlinearities from the means for scanning the AFM probe. See, e.g. FIG. 6 in Schabert et al., supra. Thus, it is often difficult to obtain registration and compare features between separate images. A further drawback to this approach is the limited scan sizes of both the AFM and confocal images. Standalone AFMs with optical lever detection, for example, have a very limited scan range without a method for optically tracking the cantilever, on the order of tens of microns. Designs with optical tracking have been introduced to circumvent this problem. However, the scan range of the independently scanned in-focus voxel is stringently limited to the field of view of the microscope objective, which is a 200 .mu.m diameter region for a 100.times. objective. Off-axis optical aberrations may even preclude the use of the entire field of view of the objective.